The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. The catalytic cycles of enzymes such as protein kinases, G proteins, small GTPases, and ATPases are crucial to the ability of these molecules to regulate themselves and other proteins. Aberrations in catalytic activity of a single enzyme can result directly in disease. Detailed understanding of the regulatory mechanisms employed by such enzymes is lacking, however, because despite intense study, the mechanism of phosphoryl transfer is still incompletely understood. Acetate kinase is an enzyme with unique catalytic properties. The study of its structure and function may provide new insights into the chemistry of enzyme-catalyzed phosphoryl group transfer. A form of the enzyme phosphorylated on a glutamyl residue has essential hallmarks of a covalent intermediate in catalysis. Yet the phosphoryl group is transferred with inversion of configuration; this change in stereochemistry is typically taken as evidence (often the sole evidence) for a direct, in-line transfer of phosphate from substrate to product without an enzyme-linked covalent intermediate. Can these two lines of evidence be reconciled? The answer to this question is critical to a general understanding enzymatic phosphoryl transfer. The long-term goal of this project is to elucidate the mechanism of acetate kinase, to rethink the mechanisms of phosphoryl transfer in other enzymes based on these results, to understand the evolution of enzyme structure and function in this family, and, eventually, to assess the ramifications of the acetate- kinase mechanism for bacterial physiology. In the proposed project period, the structure of the thermostable acetate kinase from Methanosarcina thermophila will be determined by X-ray crystallography in the presence of various combinations of substrates and inhibitors. The structure of the relatively stable covalent catalytic intermediate will be determined. Comparative structural studies with acetate kinase and butyrate kinase from other species will aid in the interpretation of active-site structures, and will provide information about the structural basis of substrate specificity and thermophily in this enzyme family. Residues critical to catalysis will be identified by inspection of the crystal structures and mutagenesis experiments; interesting mutants will be analyzed biochemically and structurally. Principles learned from the study of acetate kinase will be extended to other phosphotransferase, beginning with hexokinase. The proposed studies should provide an elucidation of the mechanism of acetate kinase, which will have implications for the understanding of phosphoryl transfer in many other enzymes.